Studies with sera from ferrets infected with single influenza viruses and from people immunized with inactivated influenza vaccines were performed by our laboratory to confirm that A/Johannesburg/33/94 (J94 [H3N2]) and B/Harbin/7/94 (B94) are markedly different from previous vaccine strains. These data were used as part of the justification of the USPHS and WHO to recommend inclusion of J94 and B94 in inactivated influenza vaccines for the 1995-1996 influenza season. Our laboratory produced reference reagents for these strains to facilitate qualification and release of approximately 70 million doses of vaccine for the United States. In addition, as a reference strain for production purposes, we developed and cloned a reassortant influenza virus (designated RESVIR 8) using A/Puerto Rico/8/34 (PR8 [H1N1] to provide increased ability to replicate in eggs combined with J94 (to provide surface glycoproteins derived from the hemagglutinin [HA] and neuraminidase [NA] genes of the wild type strains). As in the past, the undesired NA of PR8 provided a replication advantage to strains in the reassorting process. To investigate the competitive advantage of PR8 NA, our lab performed enzyme kinetic studies to compare PR8 NA with NAs from other H1N1 and H3N2 strains. The kinetic studies indicated that PR8 NA is the most efficient NA whether large or small chemical substrates are used. Since PR8 NA was as efficient in H3N1 reassortants as in PR8, HA probably has no effect on the enzyme function. The origins of the gene segments of interest from the new reassortant virus were confirmed using methods developed by our lab using polymerase chain reaction (PCR) and endonuclease restriction mapping. For several contemporary H3N2 viruses included in the studies, no DNA was obtained with specific primers based on conserved sequences from published information. In addition, predicted restriction sites were absent for some contemporary H3N2 viruses. Therefore, the DNA sequences of specific genes from influenza viruses of interest were determined by standard techniques based on PCR. The sequence studies confirmed base substitutions to explain the loss of restriction sites in the gene segments, and suggested that certain "hot spots" for base substitution may exist within a gene. The sequence data are consistent with genetic drift, and do not suggest a recent reassorting event (antigenic shift) in nature to explain genetic changes in the contemporary H3N2 strains. These studies continue in order to improve understanding of the biology of influenza viruses and the factors affecting influenza virus replication.